• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.41-51
• /
• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.1-9
• /
• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Journal of Embryo Transfer
• /
• v.18 no.2
• /
• pp.91-96
• /
• 2003

The aim of this study was conducted to evaluate the effect of sugar kinds and combination of various sugars on acrosome damage of post-thaw spermatozoa in canine. The extender used in this study was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as control (fructose, xylose, trehalose), two combinations (Fru+Tre, Fru+Xyl, Tre+Xyl) and three combinations (Fru+Tre+Xyl). The acrosome damage rate of post-thaw spermatozoa in Eosin B & Fast Green stain in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fruc+Xyl, Tre+Xyl, Fruc+Tre+Xyl (83.0$\pm$5.6 vs. 82.3$\pm$3.1%, 81.7$\pm$2.1%, 72.0$\pm$2.0%; 80.3$\pm$4.5%, 76.7$\pm$3.8%, 81.0$\pm$5.6). The motility after CASA analysis in Fru+Tre was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose(79$\pm$6 vs 75$\pm$3, 74$\pm$8, 71$\pm$11, 70$\pm$4, 66$\pm$15, 63$\pm$ 12%). However, the progressive motility after CASA analysis in Fru+Tre group was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose (67$\pm$7, 64$\pm$3, 62$\pm$6, 61$\pm$8, 60$\pm$2, 57$\pm$13, 53$\pm$10%). The results indicated that the acrosome damage & progressive motility of post-thaw spermatozoa in 70 mM Fruc+Tre (two combination) following Eosin B & Fast Green stain and CASA analysis.

• Journal of Embryo Transfer
• /
• v.20 no.3
• /
• pp.289-295
• /
• 2005

This study was performed to investigate the relationship between Heat shock protein 70 (HSP70) gene polymorphism and in vitro fertilization(IVF) embryo development in the pigs. The single strand conformation polymorphism(SSCP) genotypes from HSP70 K1, K3 and K4 PCR products were detected different patterns. In cleavage rate of oocyte fertilized in vitro, HSP70 K1-AA genotype($73.1\%$) and K1-AB genotype($62.3\%$) showed significantly higher oocyte cleavage rate than HSP70 K1-BB genotype($49.3\%$)(p<0.05). And HSP70 K3-AA genotype ($72.4\%$) and K3-AB($62.2\%$) also showed significantly higher oocyte cleavage rate than HSP70 K3-BB genotype($49.1\%$)(p<0.05). The IVF embryo development of 2-cell stage according to HSP70 genotypes of sperm and pig breeds also showed a significant difference. The number of embryos developed to 2-cell stage in Landrace(28.8) and Duroc(29.8) were significantly higher than in Yorkshire(10.9)(p<0.05). And also HSP70 K4-AB genotype group(29.6) higher than HSP70 K4-AA genotype group(10.6)(p<0.05). However, the number of embryos developed to blastocyst stage did not showed significant differences among breeds as well as HSP70 genotypes. These resrults suggest that in vitro development in porcine early embryos may be affected by HSP70 genotypes and breeds.

• Journal of Embryo Transfer
• /
• v.16 no.2
• /
• pp.117-125
• /
• 2001

Vitrification of oocytes has been applied recently fur pigs, but remains elusive. The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine oocytes. When immature follicular oocytes frozen-thawed were cultured for in vitro maturation, maturation rates to metaphase-II stage were higher in oocytes with (25%) than without (15%) cumulus cells. After In vitro fertilization of oocytes frozen-thawed, the maturation rates were also significantly (P<0.05) higher in oocytes with (41%) that than without (17%) cumulus cells. However, the penetration rates were higher in oocytes without (19%) that than with (9%) cumulus. In another experiment, porcine oocytes matured in vitro were frozen and thawed for in vitro fertilization. The penetration rates were higher than in oocytes without (35%) that than with (26%) cumulus cells. However, the proportions of oocytes dead after in vitro fertilization were significantly (P<0.05) higher in oocytes with that than without cumulus cells. On the other hand, the rates of penetration and dead oocytes at 6 h after in vitro fertilization were not significant differences between oocytes with and without cumulus cells. However, the proportions of dead oocytes with (18%) and without (16%) cumulus cells were higher than in oocytes of control group (0%). These finding indicated the possible broader application for OPS, as they demonstrated that the maturation and fertilization in vitro by frozen-thawed oocytes may be accompained by cumulus cells and culture periods according to the requirements of the survival ability after freezing of mature and immature oocytes in pigs.

• Toxicological Research
• /
• v.33 no.2
• /
• pp.107-118
• /
• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

• Korean Journal of Veterinary Research
• /
• v.23 no.2
• /
• pp.137-148
• /
• 1983

This study was made for the better information of the male reproductive system on the meat-type drake, Cherry Belly X White Golden. The epithelium of ductules of epididymal region and deferent duct were observed histologically and histochemically with the progress of their development. India-ink absorbability on the luminal epithelium was also investigated after the administration of India-ink. The results are as follows; 1. Rete testis and various round ductules in immature form appeared in epididymis within 6 weeks after hatching, and simple cuboidal and simple columnar epithelium were found in the epithelia of the ductules within 8 weeks after hatching. Larger ductules were found on epididymal surface which was in the developing stage near to the immature efferent ductule. From 10th to 20th week, various ductules appeared in epididymis, and developing form of efferent ductules were much more increased on epididymal surface. The luminal epithelium of the ductules were composed of ciliated simple columnar and pseudostratified ciliated columnar cells. At the same time, deferent duct appeared. From the 21th week, various ductules in epididymis became abruptly matured. Lumen of rete testis was lined by simple squamous or simple cuboidal epithelium, and that of efferent ductules, having many folds and being larger than any others were lined by pseudostratified ciliated columnar epithelium in which ciliated columnar cells, non-ciliated cells(clear cells) and basal cells were noted. Connecting tubules of star shaped lumen were composed of pseudostratified ciliated columnar epithelium in which ciliated columnar cells, nonciliated cells, and basal cells were observed. The luminal surface of epididymal ducts was smooth and has thick pseudostratified columnar epithelium which was composed of high columnar cells and basal cells. From 26th week after hatching, sperm pooling was started in various ductules. 2. From 4th to 10th week, simple cuboidal epithelium of deferent duct transformed to simple columnar epithelium with the progress of aging. At the basement of epithelium, clear round cells were noted. From 12th to 20th week, high columnar cells with enlongated nucleus were noted on the luminal border of deferent ducts, forming folds of pseuclostratified columnar epithelium. From 20th week, the deferent duct started to have septa in it's lumen and composed mainly of pseudostratified columnar epithelium, and round cells disappeared. From 20th week, the lumen diameter of deferent duct became wider with the progress of aging, but there was no difference among the values of lumen diameter in upper, middle, and lower part of deferent ducts. At 26th week, the pooling period of sperms in deferent ducts, the lumen diameter became rapidly widen, especially in the lower part of deferent ducts. Thickness of muscular layer of ductus deferens showed gradual growth within 24 weeks but did abrupt thickening from 26th week. 3. Saliva resistant PAS granules were dotted on the top of nucleus in efferent ductules epithelium but the amount of the granules were little in the connecting ductules's epithelium. The granules reactive to acid phosphatase were abundant in the some epithelial cells of efferent ductules and connecting ductules, especially above the nucleus of cells. The granules reactive to alkaline phosphatase were noted on the luminal border of efferent ductules. Parts of free border of efferent ductules and middle portion of deferent ducts were stained slightly by alcian blue technique. India ink granules were found mainly in the epithelium of efferent ductules but were few in that of connecting ductules.

• Korean Journal of Microbiology
• /
• v.43 no.3
• /
• pp.151-158
• /
• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Korean Journal of Environmental Biology
• /
• v.38 no.3
• /
• pp.333-342
• /
• 2020

The aim of this study was to define the toxic effects of phenanthrene (PAHs) on the fertilization and normal embryogenesis rates in the two species of sea urchin (Hemicentrotus pulcherrimus and Mesocentrotus nudus). The sperm and fertilized eggs of both sea urchin species were exposed to serial dilutions of phenanthrene for 10 min and 48 hours, respectively. The fertilization rate and normal embryogenesis rate of H. pulcherrimus and M. nudus were decreased in a concentration-dependent manner. The EC₅₀ for the fertilization rate of H. pulcherrimus and M. nudus was 17.48 mg L^-1 and 16.21 mg L^-1, and the EC₅₀ for the normal embryogenesis rate was 2.99 mg L^-1 and 0.36 mg L^-1, respectively. Between the two species, H. pulcherrimus was more sensitive to phenanthrene exposure, and 48 h normal embryogenesis was the more sensitive endpoint. Therefore, the results of this study demonstrated that the exposure of both sea urchin species to phenanthrene caused alterations in egg fertilization and the early developmental stages.

• Journal of Embryo Transfer
• /
• v.21 no.3
• /
• pp.255-262
• /
• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.